Coronary heart disease (CHD) remains the leading cause of deaths in the U.S. Eating a healthful diet high in fruits and vegetables, whole grains, and other plant-based foods and low in added sugar and red or processed meat has been identified as one key measure for the prevention of CHD. Although ingredients in these foods that mediate the health effects remain to be elucidated, accumulating data have suggested that human gut microbiota metabolizes certain compounds in the diet and subsequently produces metabolites that may exert beneficial or harmful effects on CHD risk. In this proposed research, we focus on two sets of gut microbiota metabolites, enterolignans and trimethylamine N-oxide (TMAO), that originate from precursors rich in plant- based foods and animal products, respectively. Although basic science research has demonstrated that these compounds may play a critical role in the etiology of CHD through multiple pathways, large-scale epidemiological research is needed to establish their associations with CHD risk in general populations. In addition, data are absent regarding the inter-relationship among diet, microbiome, and the circulating levels of these metabolites among humans. To narrow this scientific gap, we aim to evaluate enterolignans and TMAO in relation to CHD risk and established cardiovascular disease (CVD) risk markers, including blood lipids, C- reactive protein, and hemoglobin A1c, among men and women participating in the Health Professionals Follow-up Study (HPFS) and the Nurses' Health Study II (NHSII). These aims will be examined among 750 matched incident CHD case-control pairs in HPFS and NHSII. In a unique study of men that overlaps with the HPFS - the Men's Lifestyle Validation Study (MLVS) - we will interrogate the microbiome profiles that predict the production of the metabolites and examine the relationship between diet, microbiome, the metabolites, and the CVD risk markers in 388 men who provided 1,178 fecal samples. These studies provide an unparalleled opportunity for us to examine the aims in an efficient and comprehensive manner, since rich data and resources, including archived blood samples, gut metagenome and metatranscriptome data, dietary assessments by 7-day diet records, and other data, are available in these studies. To ensure the successful implementation of this project, we have assembled an outstanding research team comprised of well- established researchers with complementary expertise in multi-disciplinary fields, including nutrition, biomarker research, CVD epidemiology, and bioinformatics. Data to be generated from this project will provide novel evidence that links diet, gut microbiota, biologically-active metabolite, and CHD risk in men and women. The evidence will also facilitate making more focused dietary recommendations for the public health prevention and clinical intervention of coronary heart disease.